HER2 aptamer-anticancer drug complex for cancer cell chemotherapy

ABSTRACT

This invention relates to a complex for cancer cell chemotherapy and, more particularly, to a HER2 aptamer-anticancer drug complex for chemotherapy of cancer cells, which includes a nucleic acid aptamer specifically binding to HER2 and an anticancer drug linked with the nucleic acid aptamer, so that HER2-positive breast cancer cells are selectively targeted and killed.

STATEMENT REGARDING SPONSORED RESEARCH OR DEVELOMENT

This invention was made with Korean Government support of Grant No.HI12C1852, awarded by the Korea Health Industry Development Institute,funded by the Ministry of Health & Welfare, Republic of Korea.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a complex for cancer cell chemotherapyand, more particularly, to a HER2 aptamer-anticancer drug complex forchemotherapy of cancer cells, which includes a nucleic acid aptamerspecifically binding to HER2 and an anticancer drug linked with thenucleic acid aptamer, so that HER2-positive breast cancer cells areselectively targeted and killed.

2. Description of the Related Art

Breast cancer is cancer that frequently occurs in women, and manytechniques for the accurate diagnosis and treatment of breast cancer arebeing developed. Typical methods for cancer treatment include surgery,radiation therapy, and chemotherapy, one or more of which may beutilized to treat cancer. Specifically, surgery is a method of removingalmost all diseased tissue, and is very effective at removing tumorsfrom specific regions, for example, the breast, colon and skin, but isunsuitable for the treatment of tumors in some regions, such as thespine, or for the treatment of dispersed tumors. Also, radiation therapyis useful in the treatment of acute inflammatory diseases, benign ormalignant tumors, endocrine dysfunction, and allergic diseases, and istypically effective at treating malignant tumors, which are composed ofrapidly dividing cells. Such radiation therapy has defects, includingweakness or loss of function of normal tissue due thereto, as well asthe concern about skin disease on the treated regions. Particularly inchildren, in which the growth of internal organs is progressing, seriousside effects such as delayed cognitive development or bone growthdisorders may result. Also, chemotherapy is widely useful in thetreatment of breast cancer, lung cancer and testicular cancer bydisturbing the replication or metabolism of cancer cells, but suffersfrom side effects induced by systemic chemotherapy used in the treatmentof cancer. Furthermore, side effects associated with chemotherapeuticagents are generally exemplified by dose-limiting toxicity (DLT), whichdictates caution upon administration of drugs. For these reasons, sideeffects attributable to chemotherapeutic agents and radiation therapyare regarded as important problems in the treatment of cancer patients.

Meanwhile, based on reports that breast cancer patients who overexpress,as one of human epidelitLal growth factor reactors, a tyrosine kinasebonded to the surface of a cell membrane, namely HER2 (Human epidermalgrowth factor receptor 2), which is present in the cell membrane andincludes an extracellular region that binds to a ligand and anendocellular region that causes protein activation, have lowerdisease-free life expectancy and a higher recurrence rate than nolitLalpersons, HER2 was established as a representative breast cancerbiomarker. Moreover, with the goal of overcoming the side effects of theexisting therapeutic methods, targeted therapy, which attacks signalingpathways related to the proliferation of cancer or inhibits angiogenesisso that cancer cells are starved to death, is receiving attention.Hence, research into HER2-targeted breast cancer treatment methods isongoing (Korean Patent Application Publication No. 10-2013-0091750), butis still incomplete.

SUMMARY OF THE INVENTION

Accordingly, the present invention has been made keeping in mind theabove problems encountered in the related art, and the present inventionis intended to provide a HER2 aptamer-anticancer drug complex for cancercell chemotherapy, which is able to effectively kill HER2-positivebreast cancer cells.

In addition, the present invention is intended to provide a HER2aptamer-anticancer drug complex for cancer cell chemotherapy, in whichan anticancer drug may be effectively delivered to HER2-positive breastcancer cells by targeting HER2 using an aptamer.

In addition, the present invention is intended to provide a HER2aptamer-anticancer drug complex for cancer cell chemotherapy, which mayeffectively treat existing drug-resistant breast cancer.

In addition, the present invention is intended to provide a HER2aptamer-anticancer drug complex for cancer cell chemotherapy, which isforted via disulfide bonding between an aptamer and an anticancer drug,whereby the complex absorbed into cancer cells is split by glutathionepresent in the cytosol, and thus the release of the drug to non-targetregions may be inhibited.

The present invention is accomplished by exemplary embodiments asfollows.

An embodiment of the present invention provides a complex for cancercell chemotherapy, comprising a nucleic acid aptamer specificallybinding to HER2 and an anticancer drug linked with the nucleic acidaptamer.

Also, the complex for cancer cell chemotherapy according to the presentinvention may be formed via disulfide bonding between the nucleic acidaptamer and the anticancer drug.

Also, in the complex for cancer cell chemotherapy according to thepresent invention, a thiol group may be introduced to a 3′ terminal ofthe nucleic acid aptamer, and the anticancer drug may be an anticancerdrug having a thiol group, and thus the disulfide bonding may be formedbetween the nucleic acid aptamer and the anticancer drug.

Also, in the complex for cancer cell chemotherapy according to thepresent invention, the nucleic acid aptamer may have a base sequence ofSEQ ID NO:1.

Also, in the complex for cancer cell chemotherapy according to thepresent invention, the anticancer drug may include DM1.

Also, the complex for cancer cell chemotherapy according to the presentinvention may be used for the treatment of breast cancer.

Also, the complex for cancer cell chemotherapy according to the presentinvention may be absorbed into HER2-positive breast cancer cells, sothat the disulfide bonding is broken by glutathione present in thecytosol to thus isolate the anticancer drug.

Another embodiment of the present invention provides a method ofmanufacturing a complex for cancer cell chemotherapy, comprising anaptamer preparation step of preparing a nucleic acid aptamer having anaptamer base sequence specifically binding to HER2, and a complexformation step of forming an aptamer-anticancer drug complex by reactingthe aptamer prepared in the aptamer preparation step with an anticancerdrug.

Also, in the method according to the present invention, the aptamerpreparation step may include forming a nucleic acid aptamer having abase sequence of SEQ ID NO:1 specifically binding to HER2, introducing athiol group to a 3′ terminal of the nucleic acid aptamer, performing2′-O-methyl modification, and activating a 3′ thiol group by reactionwith dithiothreitol in a triethylammonium acetate buffer.

Also, in the method according to the present invention, the complexformation step may include providing DM1 dissolved in dimethylsulfoxide, and reacting the aptamer and DM1 at a ratio of 1:1000 in apotassium phosphate buffer containing dimethyl sulfoxide (DMSO) andethylenediaminetetraacetic acid (EDTA), thus forming theaptamer-anticancer drug complex.

According to embodiments of the present invention, the following effectsmay be obtained.

The present invention is effective at killing HER2-positive breastcancer cells.

Also, the present invention is effective at delivering an anticancerdrug to HER2-positive breast cancer cells by targeting HER2 using anaptamer.

Also, the present invention is effective at treating existingdrug-resistant breast cancer.

Also, according to the present invention, when the complex, which isfolioed via disulfide bonding between the aptamer and the anticancerdrug, is absorbed into cancer cells, it is split by means of glutathionepresent in the cytosol, thus effectively inhibiting the release of adrug to non-target regions.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The above and other features and advantages of the present inventionwill be more clearly understood from the following detailed descriptiontaken in conjunction with the accompanying drawings, in which:

FIG. 1 shows confocal microscopy images depicting the extent ofexpression of HER2 in different breast cancer cell lines;

FIG. 2 show confocal microscopy images depicting the targetingcapability of the complex in the BT474 breast cancer cell line;

FIG. 3 shows confocal microscopy images depicting the targetingcapability of the complex in the HER2-positive breast cancer cell lineand the HER2-negative breast cancer cell line, which are co-cultured;

FIG. 4 shows the results of quantification of the number of cells inwhich His3 is detected depending on whether HER2 is expressed or not;

FIG. 5 shows the results of measurement of the ability of the complex toselectively kill HER2-positive breast cancer cells;

FIG. 6 shows the results of measurement of the tumor volume to evaluatethe ability of the complex to selectively kill HER2-positive breastcancer cells in breast cancer disease animal models;

FIG. 7 shows images depicting the results of a TUNEL assay to evaluatethe ability of the complex to selectively kill HER2-positive breastcancer cells in breast cancer disease animal models;

FIG. 8 shows the results of measurement of the cell viability ofexisting drug-resistant cell lines;

FIG. 9 shows images depicting the results of Western blot analysis ofexisting drug-resistant cell lines;

FIG. 10 shows images depicting the results of Western blot analysis toevaluate the effects of the drug complex on existing drug-resistant celllines;

FIG. 11 shows the results of measurement of the tumor volume to evaluatethe effects of the complex on existing drug-resistant disease animalmodels; and

FIG. 12 shows an in-vivo bioluminescence image to evaluate the effectsof the complex in existing drug-resistant disease animal models.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Hereinafter, a detailed description will be given of a HER2aptamer-anticancer drug complex for cancer cell chemotherapy accordingto the present invention, with reference to the appended drawings.Unless otherwise defined, all terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thepresent invention belongs. If the meaning of the term used hereinconflicts with the general meaning thereof, it accords to the definitionused herein. In the following description of the present invention,detailed descriptions of known constructions and functions incorporatedherein will be omitted when they may make the gist of the presentinvention unclear. As used herein, when any part “includes” any element,it means that other elements are not precluded but may be furtherincluded, unless otherwise mentioned.

According to an embodiment of the present invention, a complex forcancer cell chemotherapy includes a nucleic acid aptamer specificallybinding to HER2 and an anticancer drug linked with the nucleic acidaptamer.

As used herein, the term “nucleic acid aptamer” refers tosingle-stranded DNA or RNA having high affinity and selectivity to aspecific target material. The aptamer advantageously exhibits highaffinity to a target material compared to an antibody, and high thermalstability to thus enable long-term storage at room temperature,facilitates chemical modification to thus be relatively simply producedat low cost, and may be regenerated within a short time even whendegenerated. Hence, in the present invention, a nucleic acid aptamerspecifically binding to HER2 upon formation of the complex is adopted.The nucleic acid aptamer may have various aptamer base sequences, whichmay specifically bind to HER2, but preferably has a base sequence of SEQID NO:1. In order to form a complex thereof with the anticancer drug, athiol group is introduced to the 3′ terminal of the nucleic acidaptamer.

As the anticancer drug, any drug may be used so long as it is linkedwith the nucleic acid aptamer to thus kill cancer cells. Preferablyuseful is a drug having a thiol group, and more preferably useful ismaytansine (a maytansinoid, DM1). Maytansine (DM1) is a drug which islinked to a tubulin protein for forming mitotic spindles in the G2/Mphase of a cell cycle so that the function of tubulin is inhibited toinduce the formation of abnormal mitotic spindles, thus suppressing thenormal arrangement of chromosomes, thereby preventing mitosis andleading to the apoptosis of cancer cells. The 3′ terminal of the nucleicacid aptamer is introduced with a thiol group, and as the anticancerdrug, an anticancer drug having a thiol group is used, whereby thenucleic acid aptamer and the anticancer drug are linked via disulfidebonding, thus forming a complex. Since HER2, which is overexpressed, isfound in breast cancer, the complex includes the nucleic acid aptamerfor targeting HER2 to thereby effectively target the breast cancercells. As for the complex that is formed via disulfide bonding,glutathione, which breaks disulfide bonds, is present in the cytosol butabsent from the blood and lymph, and thus the complex of the presentinvention is absorbed into breast cancer cells and then the anticancerdrug is isolated, thereby effectively releasing the drug to cancercells, rather than non-target regions, ultimately decreasing sideeffects and increasing the effect of cancer cell apoptosis.

Another embodiment of the present invention addresses a method ofmanufacturing a complex for cancer cell chemotherapy, including: anaptamer preparation step of preparing a nucleic acid aptamer having anaptamer base sequence specifically binding to HER2, and a complexformation step of forming an aptamer-anticancer drug complex by reactingthe aptamer prepared in the aptamer preparation step with an anticancerdrug.

In the aptamer preparation step, a nucleic acid aptamer having anaptamer base sequence specifically binding to HER2 is prepared.Particularly, a nucleic acid aptamer having a base sequence of SEQ IDNO:1, specifically binding to HER2, is formed, a thiol group isintroduced to the 3′ terminal of the nucleic acid aptamer (to substituteOH with SH), and 2′-O-methyl modification is performed, after which thereaction with dithiothreitol in a triethylammonium acetate buffer iscarried out, thereby activating the 3′ thiol group, yielding the nucleicacid aptamer.

In the complex formation step, the aptamer prepared in the aptamerpreparation step is reacted with the anticancer drug, thus forming theaptamer-anticancer drug complex. Particularly, DM1 dissolved in dimethylsulfoxide is provided, and the aptamer and DM1 are reacted at a ratio of1:1000 in a potassium phosphate buffer containing DMSO and EDTA, therebyobtaining the aptamer-anticancer drug complex.

Still another embodiment of the present invention addresses apharmaceutical composition for the treatment of breast cancer, includingthe above complex for cancer cell chemotherapy.

The pharmaceutical composition for the treatment of breast cancer mayinclude (a) a pharmaceutically effective amount of the above complex forcancer cell chemotherapy; and (b) a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically effective amount” may referto an amount sufficient to achieve the effect of breast cancer therapy.

In the pharmaceutical composition for the treatment of breast cancer,the pharmaceutically acceptable carrier may include those typically usedfor formulations, and examples thereof may include, but are not limitedto, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acaciarubber, calcium phosphate, alginate, gelatin, calcium silicate,microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water,syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate,talc, magnesium stearate, and mineral oil. The pharmaceuticalcomposition for the treatment of breast cancer may further include, inaddition to the above components, a lubricant, a humectant, a sweetener,a flavoring agent, an emulsifier, a suspending agent, a preservative,etc.

The pharmaceutical composition for the treatment of breast cancer may beadministered orally or parenterally (e.g. intravenous, intraperitoneal,intramuscular, subcutaneous, or topical administration).

The appropriate dose of the pharmaceutical composition for the treatmentof breast cancer may vary depending on the formulation method,administration mode, the subject's age, weight, and gender, diseaseseverity, diet, administration time, administration route, excretionrate, and response sensitivity, and doctors who are normally trained mayeasily determine and prescribe the dose effective for desired treatment.

According to a method that may be easily executed by those skilled inthe art to which the present invention belongs, the pharmaceuticalcomposition for the treatment of breast cancer may be formulatedtogether with a pharmaceutically acceptable carrier and/or excipientinto a unit dose form, or may be packed in multiple-dose containers. Assuch, the formulation may be provided in the form of a solution,suspension or emulsion in oil or an aqueous medium, or in the form of anextract, a powder, a granule, a tablet, or a capsule, or may furtherinclude a dispersant or a stabilizer.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed to limit the scope of the present invention.

EXAMPLE 1 Formation of Aptamer

(1) A nucleic acid (RNA) aptamer[5′-GGGAGGACGAUGCGGGACUGUACGGGGCUCUGUGCAGACGACUCGCCCGA-3′ (SEQ ID NO:1)]having an aptamer base sequence specifically binding to HER2 wasdesigned, and a thiol group was introduced to the 3′ terminal of thenucleic acid aptamer (to substitute OH with SH), followed by 2′-O-methylmodification, thereby increasing the stability of the nucleic acidaptamer.

(2) Thereafter, the nucleic acid aptamer was reacted at room temperaturefor 15 min with 10 μl of 1.0 M dithiothreitol (DTT) in a 0.1 Mtriethylammonium acetate (TEAA) buffer, whereby the 3′ thiol group wasactivated, and also, extraction was performed three or more times usingethyl acetate to remove excess DTT.

EXAMPLE 2 Formation of Aptamer-Snticancer Drug Complex (Aptamer-DM1)

The aptamer finally obtained in Example 1 was reacted with an anticancerdrug (DM1), thereby manufacturing an aptamer-anticancer drug complex.Particularly, DM1 was dissolved in dimethyl sulfoxide (DMSO) to give a10 mM stock, and the aptamer-DM1 complex was formed in a 100 mMpotassium phosphate buffer (pH 7.0) containing 50% DMSO and 2 mMethylenediaminetetraacetic acid (EDTA). As such, the aptamer and DM1were reacted at a ratio of 1:1000 at room temperature for 48 hr.

EXAMPLE 3 Purification of aPtamer and Aptamer-Anticancer Drug Complex

The aptamer, finally obtained in Example 1, and the aptamer-anticancerdrug complex, finally obtained in Example 2, were purified throughhigh-performance liquid chromatography (HPLC). Separation was progressedusing an Eclipse XDB-C18 column through the gradient of a binding buffer(95% 0.1 M TEAA, 5% acetonitrile) and an elution buffer (50% 0.1 M TEAA,50% acetonitrile). The peaks of HPLC were analyzed using massspectroscopy, among which only peaks that matched the molecular weightsof the aptamer and the aptamer-anticancer drug complex were selected.The aptamer (Aptamer) and the aptamer-anticancer drug complex(Aptamer-DM1), purified in Example 3, were used for the followingExamples.

Example 4 Culture of Breast Cancer Cell Lines and Evaluation ofExpression of HER2 in Breast Cancer Cell Lines

(1) Each of breast cancer cell lines, including MCF7, T47D, SKBR3,BT474, MDA-MB-453, and MDA-MB-231, was cultured under conditions of 5%CO₂ and 37° C. in a Dulbecco's modified Eagle's medium (DMEM) containing10% fetal bovine serum (FBS), streptomycin-penicillin (100 U/ml) andFungizone (0.625 μg/ml).

(2) The cells cultured on 8-well chamber slides were fixed with 4%paraformaldehyde, washed with PBS, and cultured for 10 min with 0.2%Triton X-100. Thereafter, the cells were cultured overnight at 4° C.with primary antibodies in an antibody dilution buffer and then culturedat room temperature for 2 hr with fluorescence-conjugated secondaryantibodies. Thereafter, the cells were mounted on ProLong Gold AntifadeReagent containing DAPI (4′,6′-diamidine-2′-phenylindoledihydrochloride). Particularly, each of the cultured breast cancer celllines was subjected to immunocytochemistry (ICC) using a HER2 antibody,the nucleus thereof was stained with DAPI, and HER2 (red) and nucleus(blue) regions were imaged using a confocal microscope. The results areshown in FIG. 1.

(3) As shown in FIG. 1, HER2 was highly expressed on the cell membranesof the HER2-positive breast cancer cell lines (SKBR3, BT474 andMDA-MB-453), but was hardly expressed on the Luminal type MCF7 and T47Dbreast cancer cell lines and the Basal type MDA-MB-231 cell line.

Example 5 Evaluation of Targeting Capability of Aptamer-Anticancer DrugComplex to HER2-Positive Breast Cancer Cells

(1) ICC was performed using the HER2 antibody. The HER2-positive breastcancer BT474 cell line, the nucleus of which was stained with DAPI, wastreated with a control (DMSO), DM1, Aptamer, and Aptamer-DM1 at aconcentration of 10 nM for 48 hr, and HER2 (red) and the nucleus (blue)were imaged using a confocal microscope. The results are shown in FIG.2.

(2) HER2 and a mitotic arrest marker, namely phospho-Histone H3(p-His3), were stained with ICC, and the HER2-positive breast cancerBT474 cell line and the HER2-negative breast cancer T47D cell line, thenuclei of which were stained with DAPI, were co-cultured at a ratio of1:1 and treated with a control (DMSO), DM1, and Aptamer-DM1 at aconcentration of 10 nM for 24 hr. Thereafter, the nuclei (blue), p-His3(green), and HER2 (red) were imaged using a confocal microscope. Theresults are shown in FIG. 3. The number of cells in which p-His3 wasdetected was quantified based on whether HER2 was expressed or not. Theresults are shown in FIG. 4.

(3) As shown in FIG. 2, the expression of HER2 on the cell membrane wasobserved in the control group and the DM1-treated group (B, C, E and F),and cellular internalization of HER2 from the cell membrane was observedin the groups treated with Aptamer and Aptamer-DM1 (where cellularinternalization of HER2 is represented by yellow arrows, H, I, K and L).Thereby, Aptamer and Aptamer-DM1 can be seen to specifically recognizeHER2 to thus realize cellular internalization.

(4) As shown in FIG. 3, in the control group, the cells in which HER2was expressed are deemed to be BT474 and the cells in which HER2 was notexpressed are deemed to be T47D. In the DM1-treated group, expression ofp-His3 was found regardless of whether HER2 was expressed or not, but inthe group treated with Aptamer-DM1, p-His3 was detected only in thecells in which HER2 was expressed. As shown in FIG. 4, in theDM1-treated group, the proportions of cells in which p-His3 was detectedwere not significantly different between the HER2-positive cell line andthe HER2-negative cell line. In the group treated with Aptamer-DM1, theproportion of cells in which p-His3 was detected was remarkablyincreased in the HER2-positive cell line but was considerably decreasedin the HER2-negative cell line. This is because Aptamer-DM1, whichrecognized HER2 of BT474 cells, was introduced into the cytosol, andthus mitotic arrest was induced by the isolated DM1, and simultaneously,the expression of p-His3 was increased, from which Aptamer-DM1 can beconfirmed to specifically target only HER2.

Example 6 Evaluation of Ability of Aptamer-Anticancer Drug Complex toSelectively kill HER2-Positive Breast Cancer Cells

(1) HER2-positive breast cancer cell lines (SKBR3, BT474, MDA-MB-453)and HER2-negative breast cancer cell lines (MCF7, T47D) were treatedwith a control (DMSO), DM1, Aptamer, and Aptamer-DM1 at a concentrationof 10 nM for 72 hr, after which the cells were acquired and fixed with95% ethanol containing 0.5% Tween-20 for 24 hr and cultured for 30 minwith propidium iodide (PI, 50 μg/ml) and RNase (50 μg/ml). Thereafter,the apoptosis rate (sub-G1%) of cancer cells was measured through DNAcontent analysis using flow cytometry. The results are shown in FIG. 5.

(2) Typically, the cell cycle includes, depending on the amount of DNAin cells, G1 (cell growth phase)-S (cell replication phase)-G2/M (cellmitotic phase). The induction of apoptosis is accompanied by DNAfragmentation, and thus DNA replication and division become impossible,and the amount of DNA is remarkably decreased compared to the G1 phase.The results of apoptosis are represented by the percentage of sub-G1 inthe cell cycle.

(3) As shown in FIG. 5, the control group and the group treated withAptamer had remarkably low apoptosis rates in all cell lines, but in theDM1-treated group, a relatively high apoptosis rate was induced in allcell lines. In the group treated with Aptamer-DM1, the apoptosis ratewas high in the HER2-positive cell lines but low in the HER2-negativecell lines. Also, the group treated with Aptamer-DM1 had a highapoptosis rate in the HER2-positive cell lines compared to theDM1-treated group. Thereby, Aptamer-DM1 can be confirmed to inducespecific apoptosis only in the positive breast cancer cell line in whichHER2 is expressed and to increase apoptosis through isolation of DM1 inthe cells in coincidence with the decomposition of HER2 by the cellularinternalization of HER2.

Example 7 Evaluation of Ability of Aptamer-Anticancer Drug Complex toSelectively Kill HER2-Positive Breast Cancer Cells in Breast CancerDisease Animal Model

(1) To the fourth breast fat pads of immunodeficient 6-week-old femaleBALB/c nude mice, HER2-positive BT474 cancer cells (3×10⁶ cells) andHER2-negative T47D cancer cells (3×10⁶ cells) were transplanted, afterwhich Aptamer-DM1 (1 mg/kg, 2 mg/kg, body weight) was intraperitoneallyadministered three times a week. Then, the tumor volume was measured for18 days. The results are shown in FIG. 6, and the results of a TUNELassay are shown in FIG. 7. The tumor volume (V) was measured using acaliper and calculated according to V=(length×width²)/2. In the TUNELassay, the tumors were removed from the mice, fixed with 10%neutral-buffered formalin and embedded in paraffin. The 4 μm thicktissue section was mounted on a positively charged glass slide,deparaffinized with xylene, and dehydrated through a graded alcoholseries. After antigen retrieval, the above tissue section was boiled ina citric acid buffer (pH 6.0) and immunofluorescence was performed. Insitu TUNEL was performed on the above tissue section using a TUNEL kit(Roche Applied Sciences, Penzberg, GER), and the images thereof wereobtained using a confocal scanning microscope.

(2) As shown in FIG. 6, Aptamer-DM1 significantly decreased only thegrowth of HER2-positive BT474 tumors. As shown in FIG. 7, based on theresults of apoptosis through the TUNEL assay, Aptamer-DM1 effectivelyinduced apoptosis only in the HER2-positive BT474 tumors. Thereby,Aptamer-DM1 can be confirmed to exhibit specific anticancer effectsdepending on the expression of HER2 in the animal models.

Example 8 Establishment of Existing Drug-Resistant Cell Line andEvaluation of Properties Thereof

(1) HER2-positive breast cancer BT474 cells were subcultured atintervals of 3 days using a culture medium containing 4 μg/ml ofTrastuzumab, after which resistant clones, which were continuouslyexposed to Trastuzumab for a total of 8 months, were selected, thusestablishing a Trastuzumab-resistant cell line (BT474-Tra).

(2) BT474 and BT474-Tra cell lines were treated with Trastuzumab at aconcentration of 0 to 200 μg/ml for 48 hr, after which the cellviability was measured. The results are shown in FIG. 8. For the BT474and BT474-Tra cell lines, the expression of p185HER2, p95HER2, p-HER2,p-p95HER2, HER3, p-HER3, total-AKT, p-AKT, and PARP was analyzed viaWestern blot. The results are shown in FIG. 9. Cell viability wasmeasured using a CellTiter 96* Aqueous One Solution Cell ProliferationAssay [MTS,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium],and the amount of produced formazan was determined by measuringabsorbance at 490 nm using a Spectramax Plus 384 microplate analyzer. Inthe Western blot, the above cells were lysed in a lysis buffer (30 mMNaCl, 0.5% Triton X-100, 50 mM Tris-HCl; pH 7.4) containing phosphataseand a protease inhibitor, and the supernatant was collected and then theprotein was concentrated using a Bradford protein assay kit. 30 μg ofthe protein was subjected to SDS-PAGE and electrophoresed with anitrocellulose membrane. The membrane was cultured overnight at 4° C.with primary antibodies diluted in bovine serum albumin (BSA) and thencultured with horseradish peroxidase (HRP)-conjugated rabbit IgG(1:3000-1:10,000). Signal intensity was measured using an EnhancedChemiluminescence Kit and an X-ray film, and quantified with AlphaEaseFCsoftware.

(3) As shown in FIG. 8, the Trastuzumab-resistant BT474 cell lineexhibited lower sensitivity to Trastuzumab than the BT474 cell line. Asshown in FIG. 9, in the Trastuzumab-resistant BT474 cell line, theexpression of full-length p185HER2, mutant p95HER2, and phosphorylatedactive-form p-HER2 and p-p95HER2 was considerably increased, and theexpression of p-HER3/HER3 and p-Akt was remarkably increased.

EXAMPLE 9 Evaluation of Effect of Aptamer-Anticancer Drug Complex inExisting Drug-Resistant Cell Line

(1) The Trastuzumab-resistant BT474 cell line and the JIMT-1 cell linederived from tumors of Trastuzumab-resistant patients were treated witha control (DMSO), DM1, Aptamer, and Aptamer-DM1 at a concentration of 10nM for 72 hr, after which the expression of HER2, p95HER2, p-HER2,p-p95HER2, HER3, p-HER3, total-AKT, and p-AKT was analyzed via Westernblot. The results are shown in FIG. 10.

(2) As shown in FIG. 10, in the group treated with Aptamer-DM1, comparedto the groups treated with the control, DM1, and Aptamer, the expressionof not only full-length p185HER2 and active-form phospho-HER2 (Tyr1221)but also HER family compounds, such as HER3 and phospho-HER3, AKT andphospho-Akt (Ser 43), was significantly reduced. The expression ofp95HER2, which is a mutant that causes resistance to Trastuzumab, wasstrongly inhibited in BT474-Tra cells. Thereby, the aptamer-anticancerdrug complex can be confirmed to effectively overcome drug resistance,which is regarded as problematic in the treatment of HER2-positivetumors.

Example 10 Evaluation of Effect of Aptamer-Anticancer Drug Complex inExisting Drug-Resistant Disease Animal Model

(1) To the fourth breast fat pads of immunodeficient 6-week-old femaleBALB/c nude mice, BT474-Tra cells (3×10⁶ cells) and JIMT-1 cells (3×10⁶cells) were transplanted to generate tumors, and each of Aptamer andAptamer-DM1 was intraperitoneally administered (1 mg/kg, body weight).Thereafter, the tumor volume was measured for 15 days. The results areshown in FIG. 11, and the results of in-vivo bioluminescence imaging ofJIMT-1-transplanted mice are shown in FIG. 12. In-vivo bioluminescenceimaging was performed in a manner in which the mice were anesthetizedand administered with 150 mg/kg of luciferin, after which the tumor sizeand the transition state were measured using a bioluminescence imagingsystem.

(2) As shown in FIG. 11, Aptamer-DM1 significantly reduced the tumorvolume in both of the two resistant disease animal models transplantedwith JIMT-1 and BT474-Tra. As shown in FIG. 12, luciferin intensity wasremarkably decreased in the group administered with Aptamer-DM1.Thereby, Aptamer-DM1 can be confirmed to overcome drug resistance evenin the resistant disease animal models.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

1. A complex for cancer cell chemotherapy, comprising a nucleic acidaptamer specifically binding to HER2 consisting of the nucleic acidsequence of SEQ ID NO:1 and anticancer drug DM1 linked with the nucleicacid aptamer wherein the aptamer includes a thiol group introduced atthe 3′ terminal end and DM1 includes a thiol group such that a disulfidebond is formed between the aptamer and DM1. 2-7. (canceled)
 8. A methodof manufacturing a complex for cancer cell chemotherapy, comprising: anaptamer preparation step of preparing a nucleic acid aptamer having anaptamer base sequence specifically binding to HER2; and a complexformation step of forming an aptamer-anticancer drug complex by reactingthe aptamer prepared in the aptamer preparation step with an anticancerdrug.
 9. The method of claim 8, wherein the aptamer preparation stepcomprises forming a nucleic acid aptamer having a base sequence of SEQID NO:1 specifically binding to HER2, introducing a thiol group to a 3′terminal of the nucleic acid aptamer, performing 2′ -O-methylmodification, and activating a 3′ thiol group by reaction withdithiothreitol in a triethylammonium acetate buffer.
 10. The method ofclaim 8, wherein the complex formation step comprises providing DM1dissolved in dimethyl sulfoxide, and reacting the aptamer and the DM1 ata ratio of 1:1000 in a potassium phosphate buffer containing dimethylsulfoxide (DMSO) and ethylenediaminetetraacetic acid (EDTA), thusforming the aptamer-anticancer drug complex.